1. Field of the Invention
The invention in the field of biochemistry and medicine relates to compositions comprising mutant proteins of plasminogen activator inhibitor-type 1 (PAI-1) which have the capacity to inhibit the enzyme elastase and to inhibit vitronectin (Vn)-dependent migration of cells. This invention also relates to uses of these proteins for the treatment of diseases and disorders associated with elastase activity or in which migration and migration-driven proliferation of cells have pathophysiologic consequences.
2. Description of the Background Art
1. Plasminogen Activators
Plasminogen activators (PAs) are specific serine proteinases that activate the proenzyme plasminogen, by cleavage of a single Arg-Val peptide bond, to the enzyme plasmin (Saksela O, Biochim Biophys Acta (1985) 823:35-65). Two plasminogen activators are found in mammals, tissue-type PA (tPA) and urokinase-type PA (uPA) (Saksela O et al, Annu Rev Cell Biol (1988) 4:93-126). These enzymes are thought to influence critically many biological processes, including vascular fibrinolysis (Bachmann E, Thromb Haemost (1987) 10:227-265), ovulation (Hsuch A J W et al, In: Haseltine FP et al, eds, Meiotic Inhibition: Molecular Control of Meiosis New York: Liss 1988:227-258), inflammation (Pollanen J et al., Adv Cancer Res (1991) 57:273-328), tumor metastasis (Dano K et al., Adv Cancer Res (1985) 44:139-266), angiogenesis (Moscatelli D et al., Biochim Biophys Acta (1988) 948:67-85), and tissue remodeling (Saksela, supra).
The regulation of PAs is a complex process controlled on many levels. The synthesis and release of PAs are governed by various hormones, growth factors, and cytokines (Saksela, supra; Dano et al., supra). Following secretion, PA activity can be regulated both positively and negatively by a number of specific protein-protein interactions. Activity can be enhanced or concentrated by interactions with fibrin (Hoylaerts M et al., J Biol Chem (1982) 257:2912-2919), the uPA receptor (uPAR) (Ellis V et al., Semin Thromb Hemost (1991) 17:194-200), the tPA receptor (tPAR) (Hajjar K A et al., J Biol Chem (1990) 265:2908-2916), or the plasminogen receptor (Plow E F et al., Thromb Haemost (1991) 66:32-36).
PA activity can be downregulated by specific PA inhibitors (PAIs) (Lawrence, D. A et al., In: Molecular Biology of Thrombosis and Hemostasis, Roberts, H. R. et al., (Eds.), Marcel Dekker Inc., New York, chapter 25, pp. 517-543 (1995)). In addition, PA activity is dependent on its location or microenvironment and may be different in solution (e.g., circulating blood) as compared to a solid-phase (e.g., on a cell surface or in the extracellular matrix (ECM)). The overall activity of the PA system is determined by the interactions among these various elements and the balance between the opposing activities of enzymes and inhibitors.
The PAIs have become recognized as critical regulators of the PA system. The identification of an efficient inhibitor of tPA in endothelial cells (ECs) was first reported in 1983 (Loskutoff D J et al., Proc Natl Acad Sci USA (1983) 80:2956-2960). Four kinetically relevant PAls are currently recognized:PAI type 1 (PAI-1), initially described as the endothelial cell PAI; PAI type 2 (PAI-2), also referred to as placental PAI; PAI type 3 (PAI-3), also known as activated protein C (APC) inhibitor and proteinase nexin 1 (PN-1), also called glia-derived neurite-promoting factor. The present invention is directed in particular to PA-1.
2. Other Serine Proteinases
Elastase is a serine proteinase released by activated neutrophils and macrophages and monocytes. During inflammatory responses, neutrophils are activated and release elastase leading to tissue destruction through proteolysis. In the lung, elastase degrades elastic tissues and leads to emphysema. Elastase is also a compounding factor in cystic fibrosis (CF) and in both adult and infant acute respiratory distress syndrome (ARDS). Elastase has also been implicated in TNF-mediated inflammation (Massague, J. et al., Annu. Rev. Biochem. 62:515-541 (1993) and HIV infection (Bristow, C. L. et al., International Immunol. 7:239-249 (1995)).
Elastase has a broader spectrum of reactivity than plasminogen activators each of which acts preferentially on a precursor substrate to activate it.
The natural defense to elastase is a protein called .alpha.1 anti-trypsin (.alpha..sub.1 AT) or .alpha.1 proteinase inhibitor (.alpha..sub.1 PI). Patients who are deficient in .alpha..sub.1 AT are prone to emphysema, especially smokers. Furthermore, smoking provokes inflammation. In such .alpha.1AT deficiencies, the enzyme is present (CRM.sup.+) but is functionally impaired. In addition, even in individuals with normal enzyme, smoking directly inactivates .alpha..sub.1 AT. Therefore, an improved inhibitor of elastase would be highly desirable for the prevention of emphysema in susceptible subjects or for reversal of the pathophysiological process leading to this an other related diseases.
3. Serpins
The major PAls belong to the serine proteinase inhibitor (serpin) gene superfamily which includes many proteinase inhibitors in blood as well as other proteins with unrelated or unknown function (Huber R et al., Biochemistry (1989) 28:8951-8966). The serpins share a common tertiary structure and have evolved from a common ancestor. Serpins regulate many processes including coagulation, fibrinolysis, complement activation, ovulation, angiogenesis, inflammation, neoplasia, viral pathogenesis and allergic reactivity.
Current models of serpin structure are based on x-ray crystallographic studies of one member of the family, .alpha..sub.1 AT (reviewed in Huber et al., supra). An interesting feature of the structure of a modified form of .alpha.1AT, cleaved in its reactive center (Loebermann H et al., J Mol Biol (1984) 177:531-557), is that the two amino acid residues that normally constitute the reactive center (Met-Ser bond), are found on opposite ends of the molecule, separated by almost 70 .ANG.. This is shown for PAI-1 in FIG. 2 and can be compared to the active structure modeled in FIG. 1. Relaxation of a strained configuration takes place upon cleavage of the reactive-center peptide bond, rather than a major rearrangement of the inhibitor structure. In this model, the reactive center is part of an exposed loop, also called the strained loop. Upon cleavage, this loop moves or "snaps back," becoming one of several central strands in a major .beta. sheet structure. This transformation is accompanied by a large increase in thermal stability, presumably as a result of the reconstitution of the six-stranded .beta. sheet A.
Synthetic peptides homologous to the reactive-center loops of serpins, when added in trans, incorporate into their respective molecules, presumably as a central strand of the major .beta. sheet structure and increase the thermal stability of the molecule like that observed after cleavage at the reactive center. This structural change converts the serpin from an inhibitor to a substrate for its target proteinase (Carrell R W et al., Nature (1991) 353 :576-578; Bjork I et al., J Biol Chem (1992) 267:1976-1982).
Serpins act as suicide inhibitors, reacting only once with their target proteinase to form a sodium dodecyl sulfate (SDS)-stable complex. These complexes can dissociate to yield free active enzyme together with a cleaved inhibitor similar to that seen in the .alpha.1AT crystal structure (Carrell R W et al., In: Barrett A J, et al. eds., Proteinase Inhibitors. Amsterdam: Elsevier Science Publishers 1986:403-420) and modeled in FIGS. 1 and 2 for PAI-1.
Serpins interact with their target proteinase by providing a "bait" amino acid residue in the reactive center which is thought to mimic the normal substrate of the enzyme and to associate via its side-chain atoms with the specificity crevice, or S1 site, of the enzyme (Huber et al., supra; Carrell et al., supra; Shubeita H E et al., J Biol Chem (1990) 265:18379-18385; York J D et al., J Biol Chem (1991) 266:8495-8500; Sherman P M et al., J Biol Chem (1992) 267:7588-7595). The bait amino acid is designated the P1 residue. The amino acids toward the N-terminal side of the scissile reactive-center bond are labeled in order P1, P2, P3, etc., and the amino acids on the carboxyl side are labeled P1', P2', etc. (Carrell et al., 1986, supra). The amino acid residues in the reactive center loop of PAI-1 (residues 332-351 of SEQ ID NO:3) are shown below labeled according to the foregoing naming convention. Also noted are the numerical positions in the full sequence of mature PAI-1:
332 330 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 P15 P14 P13 P12 P11 P10 P9 P8 P7 P6 P5 P4 P3 P2 P1 P1' P2' P3' P4' P5' Gly Thr Val Ala Ser Ser Ser Thr Ala Val Ile Val Ser Ala Arg Met Ala Pro Glu Glu
The complex between serpins and their target proteinases is thought to be covalently linked via an ester bond between the active-site serine residue of the proteinase and the new C-terminal end of the P1 residue, forming an acyl-enzyme complex (Lawrence D A et al., J Biol Chem (1995) 279:25309-25312). The association between inhibitor and proteinase also involves regions other than the P1 residue of the serpin and other than the catalytic site of the proteinase, based on the characterization of two recombinant PA mutants in which six or seven amino acids were deleted from the catalytic domains. These mutant PAs were almost completely refractory to inhibition by PAI-1, suggesting that the residues distant from the active site are nevertheless critical for the interaction with PAI-1 (Madison E L et al., Nature (1989) 339:721-724; Adams D S et al., J Biol Chem (1991) 266:8476-8482).
4. Plasminogen Activator Inhibitor Type 1 (PAI-1)
PAI-1 (see Table 1) is considered one of the principal regulators of the PA system. It is a single chain glycoprotein with a molecular weight of 50 kDa (Van Mourik J A et al., J Biol Chem (1984) 259:14914-14921) and is the most efficient inhibitor known of the single- and two-chain forms of tPA and of uPA (Table 1) (Lawrence D et al., Eur J Biochem (1989) 186:523-533). PAI-1 also inhibits plasmin and trypsin (Hekman C M et al., Biochemistry (1988) 27:2911-2918) and also inhibits thrombin and activated protein C, though with much lower efficiency.
TABLE 1 ______________________________________ SUMMARY OF CHARACTERISTICS OF PAI-1 2nd Order Rate Other Other Names Sources Constants (M.sup.-1 s.sup.-1) ligands ______________________________________ Endothelial In vivo uPA 9.0 .times. 10.sup.6 Vitronectin
Platelet platelets tctPA 2.7 .times. 10.sup.7 Heparin
 Fast-acting smooth sctPA 4.5 .times. 10.sup.6 Fibrin
muscle .beta.-Migrating LPS- APC 1.1 .times. 10.sup.4
activated Plasmin 6.6 .times. 10.sup.5 endothelium In vitro: Trypsin 7.0 .times. 10.sup.5 many cells Thrombin 10.sup.3 -2 .times. 10.sup.5 types ______________________________________ Abbreviations: LPS, lipopolysaccharide: sctPA. singlechain tPA: tctPA. twochain tPA: APC. activated protein C
PAI-1 is present in plasma at very low concentrations, ranging from 0 to 60 ng/ml (average of about 20 ng/ml or 0.5 nM) (Declerck P J etal., Blood (1988) 71:220-225) and a reported half-life of about 6-7 minutes (Vaughan D E et al., Circ Res (1990) 67:1281-1286). In a study comparing the clearance of two distinct forms of PAI-1 (active and latent; see below), the active form was cleared biphasically (half-lives of 6 and 25 minutes), whereas latent PAI-1 was cleared with a half-life of only 1.7 minutes (Mayer E J et al., Blood (1990) 76:1514-1520).
PAI-1 is present in platelets and other tissues and is produced by many cell types in culture (Erickson L A et al., J Clin Invest (1984) 74:1465-1472; Sawdey M S et al., J Clin Invest (1991) 88:1346-1353; Krishnamurti C et al., Semin Thromb Hemost (1992) 18:67-80). In vivo, the primary extravascular source of PAI-1 appears to be vascular smooth muscle cells (SMCs) (Loskutoff D J, Fibrinolysis (1991) 5:197-206). During endotoxemia or other pathological conditions, ECs become a major site of PAI-1 synthesis (Pyke C et al., Cancer Res (1991) 51:4067-4071; Schneiderman J et al., Proc Natl Acad Sci USA (1992) 89:6998-7002; Keeton M et al., Am J Pathol (1993) 142:59-70).
Plasma PAI-1 is present as a complex with vitronectin (Vn) or S protein (Declerck P J et al., J Biol Chem (1988) 263:15454-15461). PAI-1 is also associated with Vn in the ECM in culture and may be involved in maintaining the integrity of the cell substratum in vivo (Mimuro J et al., Blood (1987) 70:721-728; Mimuro J et al., J Biol Chem (1989) 264:5058-5063).
The major source of plasma PAI-1 is not known but is likely to be vascular SMCs, though a contribution from the platelet pool cannot be excluded. PAI-1 functions efficiently in solution and when bound to surfaces ("solid phase"), and it is likely that PAI-1 regulates fibrinolysis in both environments.
(a) PAI-1 Protein Structure and Function (See FIGS. 1-4)
PAI-1 cDNA encodes a protein of 402 amino acids that includes a typical secretion signal sequence (Ny et al., supra; Ginsburg et al., 1986, supra). Mature human PAI-1 isolated from cell culture is composed of two variants of 381 and 379 amino acids in approximately equal proportions. These two forms, likely arising from alternative cleavage of the secretion signal sequence, provide proteins with overlapping amino-terminal sequences of Ser-Ala-Val-His-His and Val-His-His-Pro-Pro (portion of SEQ ID NO:2 and 3) (Lawrence et al., 1989, supra). This latter sequence is generally referred to as mature PAI-1.
PAI-1 is a glycoprotein with three potential N-linked glycosylation sites containing between 15 and 20% carbohydrate (Van Mourik J A et al., supra). Mature PAI-1 contains no cysteine residues, facilitating efficient expression and isolation of recombinant PAI-1 from E. coli. PAI-1 produced in E. coli, although nonglycosylated, is functionally very similar to native PAI-1. Recombinant PAI-1 can be isolated from E. coli in an inherently active form (see below), which contrasts with PAI-1 purified from mammalian cell culture (Lawrence et al., 1989, supra; Hekman et al, 1988,supra).
(b) Active and Latent Conformation
PAI-1 exists in an active form as it is produced by cells and secreted into the culture medium and an inactive or latent form that accumulates in the culture medium over time (Hekman C M et al., J Biol Chem (1985) 260:11581-11587; Levin E G et al., Blood (1987) 70:1090-1098). The active form spontaneously converts to the latent form with a half-life of about 1 h at 37.degree. C. (Lawrence et al., supra; Hekman et al., supra; Levin E G et al., 1987, supra).
The latent form can be converted into the active form by treatment with denaturants, negatively charged phospholipids or Vn (Lambers et al., supra, Hekman et al., supra; Wun T-C et al, J Biol Chem (1989) 264:7862-7868). Latent PAI-1 infused into rabbits became reactivated in vivo by an unknown mechanism. The reversible interconversion between the active and latent structures, presumably due to a conformational change, is a unique feature of PAI-1 as compared to other serpins. The latent form appears to be more energetically favored.
The three-dimensional structure of the latent form of PAI-1 has been solved. In this structure the entire N-terminal side of the reactive center loop is inserted as the central strand into .beta. sheet A (FIG. 2) (Mottonen et al., supra) which explains the increased stability (Lawrence, D. A. et al., Biochemistry 33:3643-3648 (1994)) as well as the lack of inhibitory activity. The structure of active PAI-1 is still unknown. It has been proposed that the reactive center in active PAI-1 is exposed as a surface loop, in contrast to its position in the latent structure (FIG. 1).
(c) The Reactive-Center Loop (RCL)
The RCL region of PAI-1 has been the subject of extensive mutational analysis which demonstrated the importance of the P1 bait residue in inhibitor function, whereas the surrounding amino acids play a less critical role. Random mutagenesis of the P3, P2, and P1 residues and the P1 and P1' residues, respectively, clearly demonstrated that either arginine or lysine at P1 is essential for PAI-1 to function as an effective inhibitor of uPA (York et al., 1991, supra; Sherman et al., 1992, supra). Residues surrounding P1 can modulate PAI-1 inhibitor activity by up to two orders of magnitude and can alter target proteinase specificity. The P1' site is surprisingly tolerant of amino acid substitutions with the exception of proline which caused almost total loss of function. When an 18 amino acid segment of PAI-1 encompassing most of the RCL was replaced with the same region from PAI-2, antithrombin III, or a serpin consensus sequence, most of the requirements for PAI-1 specificity (apart from the P1 residue), were found to lie outside the RCL sequence. All three chimeras remained efficient inhibitors of tPA and uPA, and the antithrombin III chimera was not a significantly improved inhibitor of thrombin. Furthermore, the specific sequence of the RCL, the region inserted into .beta. sheet A in the latent PAI-1 structure (FIG. 1, see above), was not critical for the conversion between the active and latent conformations of PAI-1. Hence, loop insertion depends more on the flexibility of .beta. sheet A than on the specific amino acid residues in the loop. Finally, binding to Vn was not affected by these substitutions in the RCL. The P4' and P5' residues on the C-terminal side of the reactive-center bond have also been replaced with only a small effect on PAI-1 activity.
(d) Interactions with Vitronectin (Vn)
The adhesive glycoprotein Vn is a 72 kDa glycoprotein present in plasma at micromolar concentrations and associated with many tissues. Like PAI-1, Vn can exist in multiple conformational states. Vn is involved in a wide variety of physiological responses, including cell adhesion, complement activation, thrombosis, and plasma clearance of proteinase-inhibitor complexes (Tomasini, B. R. et al. (1991) Prog. Hemost. Thromb. 10, 269-305).
PAI-1 in plasma or in the subcellular matrix is stabilized by Vn. Vn-bound PAI-1 in solution is approximately twice as stable as unbound PAI-1. On ECM the half-life of PAI-1 can be &gt;24 h (Mimuro et al., supra). Most of the PAI-1 found in platelets appears to be latent, although this point is controversial (Lang I M et al., Blood (1992) 80:2269-2274). Platelets contain Vn (Preissner K T et al., Blood (1989) 74: 1989-1996), which could act to reactivate latent platelet PAI-1 (Wun et al., supra). Platelet PAI-1 may be a major factor in the resistance of platelet-rich thrombi to thrombolysis (Fay W P et al., Blood (1994) 84:351-356). Consistent with this, anti-PAI-1 antibodies enhance clot lysis when contacted with platelet-rich thrombi in vitro (Levi M et al., Circulation (1992) 85:305-312; Braaten J V et al., Blood (1993) 81: 12901299).
Vn is thought to localize PAI-1 to the ECM where it regulates local proteolytic activity (Mimuro et al., 1987, supra). Views concerning the interaction of PAI-1 with Vn are controversial probably due to the conformational variability of both proteins. The controversy is directed to both the nature and affinity of binding of these two molecules (Sigurdardottir O et al., Biochim Biophys Acta (1990) 1035: 56-61; Kost C et al., J Biol Chem (1992) 267: 12098-12105; Seiffert D et al., Biochim Biophys Acta (1 991) 1078:23-30; Salonen E-M et al., J Biol Chem (1 989) 264:6339-6343). Controversy also surrounds the Vn binding site for PAI-1, which has been localized to the somatomedin B domain at the N-terminus (Seiffert D et al., J Biol Chem (1991) 266:2824-2830) and to the C terminus of Vn between residues 348 and 370 (Kost et al., supra). Some of these conflicts may be explained by differences in affinity of binding of the active vs. latent form of PAI-1 with Vn and/or by differences in the relative abundance of PAI-1 conformers in various PAI-1 preparations.
Recent studies of the serpin mechanism of inhibition indicate that it follows a multi-step process that requires an exposed RCL (Shore, J. D. et al., (1994) J. Biol. Chem. 270, 5395-5398; Lawrence, D. A. et al., (1995) J Biol. Chem. 270, 25309-25312; Fa, M. et al., (1995) Biochem. 34:13833-13840, Wilczynska, M. et al., (1995) J. Biol. Chem. 270:29652-29655). Upon association with a target proteinase the serpin RCL is cleaved at its P.sub.1 -P.sub.1' bond and this is followed by a rapid insertion of the RCL into .beta.-sheet A yielding the stable serpin-proteinase complex. As shown by the present inventors (see Examples) a PAI-1 Vn binding epitope on the edge of .beta.-sheet A is sensitive to this conformational change in .beta.-sheet A, as well as to similar changes associated with conversion of PAI-1 to the latent form or cleavage in the RCL by a non-target proteinase. This sensitivity may provide a way to ensure the expression of PAI-1 activity at specific sites of action. For example, Vn is thought to localize PAI-1 to the ECM where it regulates local proteolytic activity (Mimuro et al., supra). In this situation it may be beneficial to permit only functionally active PAI-1 to bind to Vn. On a cell surface an inactive ligand it can be internalized and degrades. However, this type of regulation may not be as efficient on the less dynamic ECM. Therefore, to prevent Vn from becoming saturated with inactive forms of inhibitor, a system may have evolved that is sensitive to the conformational state of PAI-1, which is closely linked to its activity state.
In addition to stabilizing active PAI-1, Vn alters PAI-1 specificity, converting it to an efficient inhibitor of thrombin (Ehrlich et al., supra, Keijer, J. et al., Blood (1991) 78:1254-1261). Vn-bound PAI-1 has a 270-fold greater rate constant toward thrombin than does free PAI-1, dependent upon the source of the Vn. Although all forms of Vn can bind PAI-1, only Vn isolated under physiological conditions is able to stimulate PAI-1 to inhibit thrombin (Naski, M. C. et al., J Biol Chem (1993) 268:12367-12372). Vn also enhances the clearance of PAI-1-thrombin complexes by the low density lipoprotein receptor-related protein (LRP) (Stefansson, S. et al., (1996) J Biol. Chem. 271:8215). PAI-1 does not appear to contribute significantly to thrombin inhibition in plasma in vivo, although local concentrations of PAI-1 may have significant effects. Vn also stimulates the inhibition of tPA by PAI-1, but to a much less dramatic extent (Keijer et al., supra; Edelberg J M et al., J Biol Chem (1991) 266:7488-7493). Vn can partially restore the reduced inhibitory activity of PAI-1 RCL mutants toward tPA.
(e) Interactions with Thrombin
Given that PAI-1 is expressed at sites of inflammation and released from platelet granules upon activation, it may under these conditions be a relevant inhibitor of thrombin. While PAI-1 alone is a rather poor inhibitor of thrombin, PAI-1-Vn complexes have greatly augmented ability to inhibit thrombin (Naski. et al., supra). Vn is present in connective tissue extracellular matrices and released from platelets upon their activation. Thrombin-PAI-1 complexes form on endothelial cell ECM, which can be inhibited with antibodies to Vn (Ehrlich, H. J. et al., (1991) J. Cell Biol. 115, 1773-1781). While these authors speculated that the thrombin:PAI-1 interaction might promote PA activity by neutralizing PAI-1, this interaction may also mediate cellular clearance of thrombin. Such clearance would resemble that of tPA and uPA whose endocytosis and degradation via several members of the LDL receptor family are promoted after complex formation with PAI-1 (Nykjaer, A. et al., (1992) J. Biol. Chem. 267, 14543-14546 (Orth, K. et al., (1992) Proc. Natl. Acad. Sci. USA 89, 7422-7426; Stefansson, S. et al., (1995) J. Cell Sci. 108: 2361-2369).
(f) Clinical Significance of PAI-1 and its Interactions
Increased levels of circulating PAI-1 are associated with thrombotic disease, including myocardial infarction and deep vein thrombosis (Juhan-Vague I et al., Thromb Res (1984) 33:523-530; Hamsten A et al., N Engl J Med (1985) 313:1557-1563; Wiman B et al., J Lab Clin Med (1985) 105:265-270; Paramo J A et al., BMJ (1985) 291:573-574; Nilsson I M et al., BMJ (1985) 290:1453-1456; Aznar J et al., Br Heart J (1988) 59:535-541; Angles-Cano E et al., J Lab Clin Med (1993) 121-:646-653). Reduced postoperative fibrinolytic activity has been correlated with increased PAI-1 activity immediately following surgery (Kluft C et al., Scand J Clin Lab Invest (1985) 45:605-610), apparently mediated by a plasma factor that stimulates PAI-1 production and secretion from vascular ECs (Kassis J et al., Blood (1992) 80: 1758-1764). Consistent with these observations, the overproduction of PAI-1 in transgenic mice results in venous thrombosis primarily in the extremities (Erickson L A et al., Nature (1990) 346:74-76). In contrast, a prospective study found no correlation between PAI-1 levels and vascular disease (Ridker P M et al., Circulation (1992) 85:1822-1827).
Three cases of partial or complete PAI-1 deficiency have been reported in humans and were associated with abnormal bleeding. In one case, normal PAI-1 antigen was detected, but PAI-1 activity was significantly reduced (Schleef R R et al., J Clin Invest (1989) 83:1747-1752), whereas in another, both PAI-1 antigen and activity levels in plasma were markedly reduced with normal levels in platelets (Dieval J et al., Blood (1991) 77:528-532). A complete deficiency of platelet and plasma PAI-1 in a 9-year-old Amish girl was associated with a moderate bleeding disorder. The patient was homozygous for a 2 base pair insertion at the end of exon 4 of the PAI-1 gene (Fay W P et al., N Engl J Med (1992) 327:1729-1733) which results in a frameshift leading to a truncated PAI-1 protein and an unstable mRNA. The deficiency is inherited as an autosomal recessive disorder. Although heterozygous parents and siblings all had plasma PAI-1 activity and antigen in the normal range, they were consistently lower than homozygous normal family members. The lack of developmental and other abnormalities in this patient was considered surprising. The correlation of complete PAI-1 deficiency with abnormal bleeding clearly demonstrates that importance of PAI-1 in the regulation of hemostasis. Given the young age of the above patient, however, an additional important in vivo role of PAI-1 in the control of ovulation or tumor metastasis cannot yet be excluded (Pollanen et al., supra; Liu Y-X et al., Eur J Biochem (1991) 195:549-555).
(g) Clearance Receptors
The LDL receptor-related protein (LRP) is a cell surface receptor (family with four members) which acts as a general clearance receptor for a diverse set of ligands, including proteinase inhibitor complexes. For review, see Strickland, D. K. et al., FASEB J. 9:890-898 (1995)). Binding to LRP results in the uptake of PAI-1-proteinase complexes into cells and destruction in the lysosomal compartment. While LRP is found on all cells, these receptors are present at higher levels in liver and on the epithelial lining of the lungs.
(h) Cell Migration
Cell migration is a tightly controlled process which depends on the coordination of many factors. Migrating cells and cells with invasive phenotypes express high levels of uPA. Processes such as angiogenesis and metastasis can be blocked by proteinase inhibitors. Inactivation of the gene for uPA in mice prevents arterial stenosis due to neointima formation following vascular trauma (Carmeliet, P. et al., Circulation 90:1-144 (1994)). During wound healing vascular cells exhibit an increase in the expression of the Vn receptor (VnR) integrin .alpha..sub.V .beta..sub.3 (Liaw L et al., Circ Res 77:665-72 (1995)). VnR permits cell motility on matrix proteins deposited at the wound. Specifically, migration into the wound area is facilitated by Vn which is deposited at the site by activated platelets or derived from plasma. Migrating vascular cells also show elevated expression of uPA and its receptor uPAR which co-localize with the VnR at focal contacts. As previously understood in the art, the PAs were thought to activate a generalized proteolytic cascade resulting in matrix destruction necessary for cellular migration and invasion. However, results obtained by the present inventors and presented herein suggest a more subtle role for PAs in regulating the expression of cryptic cell attachment sites.